1. Field of the Invention
The present invention relates to a cyclic depsipeptide synthetase and a gene thereof, and a mass production system for the cyclic depsipeptide. More specifically, the present invention relates to an enzyme for synthesizing substance PF1022 having anthelmintic activity and a gene thereof, and a mass production system for the substance PF1022.
2. Description of the Related Art
The substance PF1022 [cyclo(D-lactyl-L-N-methylleucyl-D-3-phenyllactyl-L-N-methylleucyl-D-lactyl-L-N-methylleucyl-D-3-phenyllactyl-L-N-methylleucyl)] is a cyclic depsipeptide which is produced by the filamentous fungus strain PF1022 (Mycelia sterilia, FERM BP-2671), which belongs to Agonomycetales, and has an extremely high anthelmintic activity against animal parasitic nematodes (Japanese Patent Application Laid-open No. 35796/1991; Sasaki, T. et al., J. Antibiotics, 45, 692, 1992). Accordingly, this substance is useful as a anthelmintic and also as a raw material for synthesizing a highly active derivative of this substance.
Generally, the amount of secondary metabolites produced by microorganisms isolated from nature is very small. Accordingly, in order to use the secondary metabolites industrially, it is necessary to improve the amount of the production. For this purpose, the culture method and the medium composition are investigated, fermentation conditions are improved by addition of precursors and the like, and strains are improved by mutation with UV irradiation or mutation inducers. Recently, in addition to these means, genetic recombination technology has become available to improve the productivity.
For example, enhancement of expression of an enzyme gene for biosynthesis, enhancement of expression of a regulatory gene for biosynthesis, and interruption of unnecessary biosynthesis pathways have been carried out (Khetan, A. and Hu, W.-S., Manual of Industrial Microbiology and Biotechnology, 2nd edition, p. 717, 1999). Furthermore, a known particular example is a method for increasing productivity in which a hemoglobin gene of a microorganism is expressed in order to enhance oxygen utilization ability (Minas, W. et al., Biotechnol. Prog., 14, 561, 1998).
In improving productivity using gene recombination technology, the most common technique is augmentation of expression of an enzyme gene for biosynthesis. To apply this technique, it is essential that a method of transforming a microorganism has been established, that a promoter and a terminator utilizable for expression augmentation are available, that the biosynthesis pathway has been elucidated, and that the related genes have been isolated. As to the substance PF1022-producing microorganism, a foreign gene has been successfully introduced by transformation (WO97/00944); however, the gene for biosynthesis has not been isolated.
The substance PF1022 comprises a structure in which L-N-methylleucine, D-lactic acid and D-phenyllactic acid are bonded via ester bonds and amide bonds. In a producing microorganism, it is synthesized by a certain kind of a peptide-synthesizing enzyme from four molecules of L-leucine, two molecules of D-lactic acid, and two molecules of D-phenyllactic acid. Peptide-synthesizing enzymes are those which carry out biosynthesis of secondary metabolites of microorganisms, such as peptides, depsipeptides, lipopeptides, and peptide lactone, using amino acids and hydroxy acids as a substrate. Sequences of some peptide-synthesizing enzymes have been already elucidated (Marahiel, M. A. et al., Chem. Rev., 97, 2651, 1997). The reaction by this type of enzyme is entirely different from that in a system of synthesizing a protein by a ribosome using mRNA as a template. It is thought that a peptide-synthesizing enzyme has one domain for each substrate and each substrate is activated by ATP in this domain and bonded via phosphopantothenic acid in the domain, and these then form amide bonds or ester bonds by a catalytic action in the regions between each domain.